Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways

Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.     

Proteomic Analysis of Bifidobacterium longum subsp. infantis Reveals the Metabolic Insight on Consumption of Prebiotics and Host Glycans

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.     

Several archaeal homologs of putative oligopeptide-binding proteins encoded by Thermotoga maritima bind sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the beta-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), beta-1,4-mannobiose; and TM1226 (ManD), beta-1,4-mannobiose, beta-1,4-mannotriose, beta-1,4-mannotetraose, beta-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), alpha-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.     

In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded by Thermotoga maritima Bind Sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the β-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), β-1,4-mannobiose; and TM1226 (ManD), β-1,4-mannobiose, β-1,4-mannotriose, β-1,4-mannotetraose, β-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), α-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.     

An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-N-tetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves α2-6 and α2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.     

Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis

Human milk contains high amounts of complex oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-D-glucosamine is a building block of these oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-β-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human milk oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcβ1-3 linkage found in lacto-N-tetraose, the most abundant human milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcβ1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast milk. Bifidobacterium infantis N-acetyl-β-D-hexosaminidases were induced during early growth in vitro on human milk oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human milk oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.     

Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.     

Human milk oligosaccharides: every baby needs a sugar mama

Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in and unique to human milk. Originally, HMOs were discovered as a prebiotic "bifidus factor" that serves as a metabolic substrate for desired bacteria and shapes an intestinal microbiota composition with health benefits for the breast-fed neonate. Today, HMOs are known to be more than just "food for bugs". An accumulating body of evidence suggests that HMOs are antiadhesive antimicrobials that serve as soluble decoy receptors, prevent pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections. In addition, HMOs may modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, lower the risk for necrotizing enterocolitis and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition. Most data, however, stem from in vitro, ex vivo or animal studies and occasionally from association studies in mother-infant cohorts. Powered, randomized and controlled intervention studies will be needed to confirm relevance for human neonates. The first part of this review introduces the pioneers in HMO research, outlines HMO structural diversity and describes what is known about HMO biosynthesis in the mother's mammary gland and their metabolism in the breast-fed infant. The second part highlights the postulated beneficial effects of HMO for the breast-fed neonate, compares HMOs with oligosaccharides in the milk of other mammals and in infant formula and summarizes the current roadblocks and future opportunities for HMO research.     

Adhesion of Bifidobacterium spp. to human intestinal mucus

Twenty-four Bifidobacterium strains were examined for their ability to bind to immobilized human and bovine intestinal mucus glycoproteins. Each of the tested bacteria exhibited its characteristic adhesion to human and bovine fecal mucus. No significant differences were found among the taxonomic species. Among the tested bacteria, B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum adhered to human fecal mucus better than bovine fecal mucus, while the binding of B. animalis and B. lactis was not preferential. These results suggest that the mucosal adhesive properties of bifidobacteria may be a strain dependent feature, and the mucosal binding of the human bifidobacteria may be more host specific.     

Detection of a high affinity binding site in recombinant <Emphasis Type="Italic">Aleuria aurantia</Emphasis> lectin

Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.     

The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.     

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

Heparin and HS (heparan sulfate) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS binding to VEGF (vascular endothelial growth factor) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that the VEGF binding affinity likely depends on the specific structural features of these oligosaccharides, including their degree of sulfation, sugar-ring stereochemistry and conformation. Notably, the unique 3-O-sulfo group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue-specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.     

Binding and precipitation of lectins from Erythrina indica and Ricinus communis (agglutinin I) with synthetic cluster glycosides

We recently reported that tri- and tetraantennary complex type oligosaccharides with nonreducing terminal galactose residues and the triantennary asialofetuin glycopeptide can bind and precipitate certain galactose specific lectins (L. Bhattacharyya, and C.F. Brewer (1986) Biochem. Biophys. Res. Commun. 141, 963-967; L. Bhattacharyya, M. Haraldsson, and C.F. Brewer (1988) Biochemistry 27, 1034-1041). The present study investigates the binding interactions of two of these lectins, those from Erythrina indica and Ricinus communis (Agglutinin I), with mono-, bi-, and triantennary synthetic cluster glycosides, which have little structural resemblance to complex type oligosaccharides other than they possess nonreducing terminal galactose residues (R.T. Lee, P. Lin, and Y.C. Lee (1984) Biochemistry 23, 4255-4261). The enhanced affinities of the bi- and triantennary glycosides relative to the monoantennary glycoside for the two lectins are consistent with an increase in the probability of binding due to multiple binding residues in the multiantennary glycosides. The triantennary glycoside is capable of precipitating the two lectins, and quantitative precipitation data indicate that it is a trivalent ligand. The results show that the binding and precipitation activities of complex type oligosaccharides with these lectins is due solely to the presence of multiple terminal galactose residues and not to the overall structures of the oligosaccharides.     

Nucleic acid binding characteristics of group A/B hnRNP proteins

hnRNP proteins are primarily defined by their specific sedimentational, reconstitutional, and extraction properties and are presumed to be RNA binding. However, it is not clear whether all these proteins have RNA binding capabilities. Recently, using two monoclonal antibodies, fA12 and AC88, we reported that the abundance of a subclass of the highly basic A/B hnRNP proteins was specifically down regulated during terminal differentiation of human and murine cells in vitro. In this report we have examined the nucleic acid binding characteristics of this subclass and other members of the A/B hnRNP proteins in vitro. All members of class A/B hnRNP proteins appear to have similar but not identical nucleic acid binding characteristics. However, the subclass of proteins recognized by AC88 and fA12 exhibit stronger binding affinities and are shown to be highly selective in their binding to RNA vs DNA in vitro. These proteins also preferentially bind poly(U) RNA, suggesting that in vivo they may bind effectively to uridine rich motifs critical in pre-mRNA processing.     

Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary.

Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.